CN113214396B

CN113214396B - Single-chain antibody of anti-TIM 3 and application thereof in preparing medicine for treating tumor

Info

Publication number: CN113214396B
Application number: CN202010760969.4A
Authority: CN
Inventors: 张伟; 江涛; 翟优; 李冠璋
Original assignee: Beijing Neurosurgical Institute
Current assignee: Beijing Neurosurgical Institute
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2022-04-19
Anticipated expiration: 2040-07-31
Also published as: EP4174089A1; CN113214396A; US20230295294A1; WO2022022716A1

Abstract

The disclosure relates to a single-chain antibody of anti-TIM 3, and the amino acid sequence of the single-chain antibody of anti-TIM 3 comprises a sequence shown in SEQ ID NO. 1. T lymphocytes expressing single chain antibodies against TIM3 are effective in killing tumor cells.

Description

Single-chain antibody of anti-TIM 3 and application thereof in preparing medicine for treating tumor

Technical Field

The disclosure relates to the field of medical biotechnology, in particular to a chimeric antigen receptor targeting TIM3, a coding nucleic acid, an expression vector, a cell, a pharmaceutical composition and application thereof.

Background

Chimeric Antigen Receptors (CARs) are artificially synthesized T cell receptors consisting of an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The antigen binding domain is located outside the T cell membrane and includes a single chain antibody or ligand for specifically binding a target antigen. Intracellular signaling domains are located within the membrane of T cells and serve to signal the interior of the T cell to stimulate the T cell to generate an immune response.

The CAR can target a target antigen that recognizes the surface of the tumor cell, and thus, the T cell expressing the CAR can be used to target kill the tumor cell. However, existing T cells expressing the chimeric antigen receptor CAR still have poor killing ability against tumor cells.

Disclosure of Invention

The purpose of the present disclosure is to overcome the problem that the killing ability of existing T cells expressing a chimeric antigen receptor CAR to tumor cells is still weak, and to provide a single-chain antibody against TIM 3.

In order to achieve the above object, in a first aspect, the present disclosure provides a single-chain antibody against TIM3, the amino acid sequence of the single-chain antibody against TIM3 including the sequence shown in SEQ ID No. 1.

In a second aspect, the present disclosure provides a nucleic acid encoding a single chain antibody against TIM3 of the first aspect; preferably, the nucleic acid has the nucleotide sequence shown in SEQ ID NO. 2.

In a third aspect, the present disclosure provides a fusion protein comprising, in order, an antigen binding domain, a transmembrane domain, and an intracellular signaling domain; the amino acid sequence of the intracellular signaling domain comprises the amino acid sequence of the single chain antibody against TIM3 of the first aspect;

preferably, the antigen binding domain comprises a single chain antibody against CD44 and/or a single chain antibody against CD133, and the intracellular signaling domain comprises a single chain antibody against TIM 3;

more preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO. 3.

In a fourth aspect, the present disclosure provides a fusion nucleic acid encoding the fusion protein of the third aspect;

preferably, the fusion nucleic acid has the nucleotide sequence shown in SEQ ID NO. 4.

In a fifth aspect, the present disclosure provides an expression vector having inserted therein an expression cassette comprising a first nucleic acid fragment encoding an antigen binding molecule and a second nucleic acid fragment encoding an intracellular signaling molecule comprising the single chain antibody against TIM3 of the first aspect, wherein an IRES element or 2A peptide encoding sequence is inserted between the first nucleic acid fragment and the second nucleic acid fragment;

preferably, the expression cassette is a fusion nucleic acid according to the fourth aspect.

In a sixth aspect, the present disclosure provides a cell expressing a chimeric antigen receptor comprising the single-chain antibody against TIM3 of the first aspect transfected with an expression vector of the fifth aspect.

Optionally, the host cell is a T cell.

In a seventh aspect, the present disclosure provides use of a single chain antibody against TIM3 of the first aspect, a nucleic acid of the second aspect, a fusion protein of the third aspect, a fusion nucleic acid of the fourth aspect, an expression vector of the fifth aspect, a cell expressing a chimeric antigen receptor of the sixth aspect in the preparation of a medicament for treating a tumor.

Optionally, the tumor is a brain glioma.

In an eighth aspect, the present disclosure provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the cells expressing the anti-TIM 3 chimeric antigen receptor according to the sixth aspect.

Through the technical scheme, the single-chain antibody of the anti-TIM 3 provided by the disclosure can effectively improve the killing capacity of the T lymphocyte on the tumor cell, so that the T lymphocyte expressing the single-chain antibody of the anti-TIM 3 can effectively kill the tumor cell.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 is a graph of flow cytometry detection results of CAR-T1 cells provided by embodiments of the disclosure;

figure 2 is a graph of flow cytometry detection results of CAR-T2 cells provided by embodiments of the present disclosure;

figure 3 is a graph of flow cytometry detection results of CAR-T3 cells provided by embodiments of the disclosure.

Detailed Description

The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

The first aspect of the present disclosure provides a single-chain antibody against TIM3, wherein the amino acid sequence of the single-chain antibody against TIM3 comprises the sequence shown in SEQ ID No. 1.

Wherein, the sequence shown in SEQ ID NO.1 is as follows:

QVQLQQSGPGLVKPSHTLSLTCTVSGGSISSGGYYWSWTRQHPGMGLEWIGYISYSGSIYYTPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCASLDSWGSNRDYWGQGTLVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK。

t cell immunoglobulin mucin-3 (TIM3) is an important tumor immunity detection point, is widely expressed on tumor infiltrating T lymphocytes, and can inhibit the tumor immunity activity of the tumor infiltrating T lymphocytes. The single-chain antibody of anti-TIM 3 designed aiming at TIM3 can effectively block a TIM3 pathway in T lymphocytes, improve the tumor immune activity of the T lymphocytes and enhance the lethality of the T lymphocytes to the tumor cells.

The inventors of the present disclosure found that T lymphocytes expressing the above-described single-chain antibody against TIM3 were able to kill tumor cells efficiently.

A second aspect of the disclosure provides a nucleic acid encoding a single chain antibody against TIM3 of the first aspect; preferably, the nucleic acid has the nucleotide sequence shown in SEQ ID NO. 2.

Wherein, the nucleotide sequence shown in SEQ ID NO.2 is as follows:

caggtgcagctacagcagtcgggcccaggactggtgaagccttcacacaccctgtccctcacctgcactgtctctggtggctccatcagcagtggtggttactactggagttggacccgtcagcacccagggatgggcctggagtggattggatacatctcttacagtgggagtatctattacactccgtccctcaagagtcgacttaccatatcagtggacacgtctaagaaccagttctccctgaagctgagctctgtgactgccgcggacacggccgtatattactgtgcgagtttggattcctggggatctaaccgtgactactggggccagggaaccctggtcaccgtctcgagtggaggtggtggatccgaaattgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgctcactttcggcggagggaccaaggtggagattaag。

a third aspect of the present disclosure provides a fusion protein comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain connected in sequence; the amino acid sequence of the intracellular signaling domain comprises the amino acid sequence of a single chain antibody against TIM3 of the first aspect. Preferably, the antigen binding domain comprises a single chain antibody against CD44 and/or a single chain antibody against CD133 and the intracellular signaling domain comprises a single chain antibody against TIM 3. More preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO. 3.

The fusion protein shown in SEQ ID NO.3 consists of a single-chain antibody of anti-CD 44, a single-chain antibody of anti-CD 133, a single-chain antibody of CD28, CD3, T2A and a single-chain antibody of anti-TIM 3.

Wherein the amino acid sequence shown in SEQ ID NO.3 is as follows:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGL EWVAVIWYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRSDYRGYYGMDVWGQGTTVTVSSGSTSGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCRASQSVINYLAWYQQKPGQAPRLLIYDASNRASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRRNWPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDFEMHWVRQAPGQGLEWMGDIDPGTGDTAYNLKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCALGAFVYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDVVMTQSPLSLPVTPGEPASISCRSSQSLANSYGNTYLSWYLQKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHQPYTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMTNKCLLQIALLLCFSTTALSQVQLQQSGPGLVKPSHTLSLTCTVSGGSISSGGYYWSWTRQHPGMGLEWIGYISYSGSIYYTPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCASLDSWGSNRDYWGQGTLVTVSSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK。

a fourth aspect of the present disclosure provides a fusion nucleic acid encoding the fusion protein of the third aspect. Preferably, the fusion nucleic acid has the nucleotide sequence shown in SEQ ID NO. 4.

Wherein, the nucleotide sequence shown in SEQ ID NO.4 is used for coding the amino acid sequence shown in SEQ ID NO. 3.

Wherein, the nucleotide sequence shown in SEQ ID NO.4 is as follows:

caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaattctatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggagaagtgactacaggggctactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcaggcagtactagcggtggtggctccgggggcggttccggtgggggcggcagcagcgaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttatcaactacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggcctctggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtcgcaactggccgctcactttcggcggagggaccaaggtggagatcaaaggaggtggtggatccggaggtggtggctccggaggtggtggatcccaggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccgactttgaaatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggagatattgatcctggaactggtgatactgcctacaatctgaagttcaagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgaggtctgacgacacggccgtgtattactgtgcgttgggggcctttgtttactggggccagggaaccctggtcaccgtctcctcaggcagtactagcggtggtggctccgggggcggttccggtgggggcggcagcagcgatgttgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagtcttgcaaacagttatgggaacacctatttgtcttggtacctgcagaagccagggcagtctccacagctcctgatctatgggatttccaacagattttctggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggacgttggggtttattactgcttacaaggtacacatcagccgtacacgtttggccaggggaccaagctggagatcaaaaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcccgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgggagggaaggggttctctcctgacctgcggcgatgtggaggagaaccccggacccatgaccaacaagtgtctcctccaaattgctctcctgttgtgcttctccactacagctctttcccaggtgcagctacagcagtcgggcccaggactggtgaagccttcacacaccctgtccctcacctgcactgtctctggtggctccatcagcagtggtggttactactggagttggacccgtcagcacccagggatgggcctggagtggattggatacatctcttacagtgggagtatctattacactccgtccctcaagagtcgacttaccatatcagtggacacgtctaagaaccagttctccctgaagctgagctctgtgactgccgcggacacggccgtatattactgtgcgagtttggattcctggggatctaaccgtgactactggggccagggaaccctggtcaccgtctcgagtggaggtggtggatccgaaattgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgctcactttcggcggagggaccaaggtggagattaag。

a fifth aspect of the present disclosure provides an expression vector having inserted therein an expression cassette comprising a first nucleic acid fragment encoding an antigen binding molecule and a second nucleic acid fragment encoding an intracellular signaling molecule comprising a single chain antibody against TIM3 of the first aspect, the first nucleic acid fragment and the second nucleic acid fragment having inserted therebetween an IRES element or a 2A peptide coding sequence; preferably, the expression cassette is a fusion nucleic acid according to the fourth aspect.

A sixth aspect of the present disclosure provides a cell expressing a chimeric antigen receptor comprising the single-chain antibody against TIM3 of the first aspect transfected with an expression vector of the fifth aspect. Optionally, the host cell is a T cell.

A seventh aspect of the disclosure provides a use of a single chain antibody against TIM3 of the first aspect, a nucleic acid of the second aspect, a fusion protein of the third aspect, a fusion nucleic acid of the fourth aspect, an expression vector of the fifth aspect, a cell expressing a chimeric antigen receptor of the sixth aspect in the preparation of a medicament for treating a tumor. Optionally, the tumor is a brain glioma.

An eighth aspect of the present disclosure provides a pharmaceutical composition, an active ingredient of which comprises the cell expressing the anti-TIM 3 chimeric antigen receptor according to the sixth aspect.

The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.

Example 1

This example illustrates the construction of an expression vector.

(1) Nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5 are respectively synthesized by adopting a complete sequence synthesis method. The nucleotide sequence shown in SEQ ID NO.4 is used for coding the fusion protein shown in SEQ ID NO. 3; the nucleotide sequence shown in SEQ ID NO.5 is used for coding the fusion protein shown in SEQ ID NO. 6. Wherein, the fusion protein shown in SEQ ID NO.3 and SEQ ID NO.6 comprises the following components:

g1: the fusion protein shown in SEQ ID NO.3 consists of a single-chain antibody against CD44, a single-chain antibody against CD133, a single-chain antibody against CD28, CD3, T2A and a single-chain antibody against TIM 3;

g2: the fusion protein shown in SEQ ID NO.6 consists of a single-chain antibody against CD44, a single-chain antibody against CD133, CD28 and CD 3.

Wherein, the nucleotide sequence shown in SEQ ID NO.5 is as follows:

caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaattctatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggagaagtgactacaggggctactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcaggcagtactagcggtggtggctccgggggcggttccggtgggggcggcagcagcgaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttatcaactacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggcctctggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtcgcaactggccgctcactttcggcggagggaccaaggtggagatcaaaggaggtggtggatccggaggtggtggctccggaggtggtggatcccaggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccgactttgaaatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggagatattgatcctggaactggtgatactgcctacaatctgaagttcaagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgaggtctgacgacacggccgtgtattactgtgcgttgggggcctttgtttactggggccagggaaccctggtcaccgtctcctcaggcagtactagcggtggtggctccgggggcggttccggtgggggcggcagcagcgatgttgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagtcttgcaaacagttatgggaacacctatttgtcttggtacctgcagaagccagggcagtctccacagctcctgatctatgggatttccaacagattttctggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggaggctgaggacgttggggtttattactgcttacaaggtacacatcagccgtacacgtttggccaggggaccaagctggagatcaaaaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcccgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg。

the amino acid sequence of the fusion protein shown in SEQ ID NO.6 is as follows:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRSDYRGYYGMDVWGQGTTVTVSSGSTSGGGSGGGSGGGGSSEIVLTQSPATLSLSPGERATLSCRASQSVINYLAWYQQKPGQAPRLLIYDASNRASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRRNWPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDFEMHWVRQAPGQGLEWMGDIDPGTGDTAYNLKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCALGAFVYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDVVMTQSPLSLPVTPGEPASISCRSSQSLANSYGNTYLSWYLQKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHQPYTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

(2) the 2 lentiviral expression vectors of this example were obtained by inserting the 2 nucleotide sequences synthesized in step (1) separately by a conventional method using pLVX-IRES-. DELTA.NGFR (available from Clontech, Inc., cat. No. 631982) as a vector.

Example 2

This example serves to illustrate the preparation and detection of T cells expressing a chimeric antigen receptor.

(1) The 2 lentiviral expression vectors constructed in example 1 and the empty pLVX-IRES-. DELTA.NGFR vector were each packaged into a lentivirus, and then T cells were cultured, transfected and amplified in vitro according to the following methods, respectively.

(2) T cells were isolated from blood as follows: 1mL of sterile PBS and 1mL of blood are mixed uniformly, then slowly added into the upper layer of the lymphocyte separation fluid Ficoll, and centrifuged for 30min at the temperature of 4 ℃ and the speed of 400g, and the speed of acceleration and deceleration is respectively set to be 0. After centrifugation, removing upper plasma, sucking middle leucocyte layer cells, adding PBS for heavy suspension washing, and centrifuging for 10min under the condition of 100g, and accelerating and decelerating normally. After centrifugation, the supernatant was removed, 1mL of 1640+ 10% FBS + 1% double antibody +1 XGlutamine medium was added to resuspend the cells, and then stimulated to amplify using anti-human CD3/CD28 magnetic beads (purchased from Thermo Fisher Co., Ltd.), wherein the cell concentration after resuspension was 1 g10⁶cell/mL, the amount of the added magnetic beads is 100. mu.L, then 100IU/mL rhIL-2(Peprotech) is added, and the mixture is stimulated and cultured for 2 days to obtain T cells.

(3) Lentiviral transfection was performed as follows: and (3) taking 3 parts of the separated T cells, respectively adding the lentivirus packaged in the step (1), then adding polybrene with the final concentration of 6 mu g/mL, uniformly mixing, and centrifuging for 100min at the temperature of 32 ℃ and the concentration of 800 g. After the centrifugation is finished, putting the mixture into an incubator to continue culturing for 24 hours. After the culture was completed, the culture was centrifuged at 1500rpm for 15min, and the centrifuged cells were washed at 1X 10⁶The density of/mL is inoculated in a culture plate, the culture is stimulated by rhIL2-100IU/mL, and the liquid is changed every 2-3 days until 2-4 weeks, so that transfected T lymphocytes CAR-T1, CAR-T2 and CAR-T3 are obtained. Wherein, CAR-T1 is transfected with a nucleotide sequence shown in SEQ ID NO.4 and can express a fusion protein shown in SEQ ID NO. 3; CAR-T2 is transfected with a nucleotide sequence shown as SEQ ID NO.5 and can express a fusion protein shown as SEQ ID NO. 6; CAR-T3 was transfected with an empty vector.

After the culture was completed, the cells were resuspended in PBS and the proportion of the 3 CAR-T cells and the expression of the surface CAR protein were examined by flow cytometry. The detection method comprises the following steps: and respectively centrifugally collecting transfected T cells to be detected, washing with PBS for 1 time, then discarding the supernatant, adding a corresponding detection amount of monoclonal antibody according to an antibody specification, keeping out of the sun for 30min, washing with PBS, resuspending, passing through a membrane, and then detecting by a sandwich method by using a flow cytometer, wherein the antibody used in detection is a mixture of His-tag labeled CD44 and PE labeled anti-His-tag antibody. The results are shown in FIGS. 1 to 3.

As can be seen from FIGS. 1 and 2, other cells distinct from normal T lymphocytes were detected in both CAR-T1 cells and CAR-T2 cells; as can be seen in FIG. 3, the CAR-T3 cells did not detect other cells that differed from normal T lymphocytes. This indicates that the CAR-T cells transfected with the fusion gene in this example all have successfully expressed the fusion protein of interest.

Example 3

This example was used to verify the killing ability of CAR-T cells constructed in example 2 against tumor cells.

The 3 CAR-T cells transfected and cultured in example 2 were taken separately and mixed with CD44 and CD133 positive glioma stem cells GSC20, respectively, at different effective target ratios (number of T cells: number of glioma stem cells). Culturing the mixed cells in a 96-well plate, wherein each well contains 4 × 10 glioma stem cells⁴Each well of the reaction system was 200. mu.L. The culture conditions include: 37 ℃ and 5% CO₂Incubate for 4 hours at saturated humidity.

Determination of lactate dehydrogenase Activity: after the centrifugation is finished, 100 mu L of supernatant is absorbed by each hole and placed in a 96-hole enzyme label plate, and simultaneously 100 mu L of LDH substrate is added into each hole, and the reaction is carried out for 30min at room temperature in a dark place. After the reaction was completed, 50. mu.L of stop buffer was added to each well to terminate the enzymatic reaction. Optical density values (OD) were measured at 490nm in a microplate reader. The mean Optical Density (OD) values for each group were calculated and the lysis rate of each T cell on glioma stem cells was calculated as follows. The results are shown in Table 1.

The lysis rate%

TABLE 1

As can be seen from table 1, the single-chain antibody against TIM3 provided by the present disclosure can effectively enhance tumor immune activity of T lymphocytes, and enhance lethality of T lymphocytes to tumor cells.

Comparative example

The conventional second generation CAR-T cells targeting the classical tumor target EGFR vIII (EGFR vIII-CD28-CD3) were used to replace the T cells in example 3, and then the killing rate of this CAR-T on glioma stem cells GSC20 at different effective target ratios was tested as in example 3. The results are shown in Table 2.

TABLE 2

As can be seen from table 2, traditional second generation CAR-T cells targeting the classical tumor stem cell target EGFR vIII do not have the killing power on glioma stem cells as does T cells expressing single chain antibodies against TIM3 as provided by the present disclosure.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Sequence listing

<110> Neuko department of neurosurgery research in Beijing

<120> single-chain antibody against TIM3 and application thereof in preparing drugs for treating tumors

<130> 17157BJNI-LGZ

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 232

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser His

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Thr Arg Gln His Pro Gly Met Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Ile Tyr Tyr Thr Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ser Leu Asp Ser Trp Gly Ser Asn Arg Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile Val

115 120 125

Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala

130 135 140

Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp

145 150 155 160

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala

165 170 175

Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser

180 185 190

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe

195 200 205

Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu Thr Phe Gly

210 215 220

Gly Gly Thr Lys Val Glu Ile Lys

225 230

<210> 2

<211> 696

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caggtgcagc tacagcagtc gggcccagga ctggtgaagc cttcacacac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ttggacccgt 120

cagcacccag ggatgggcct ggagtggatt ggatacatct cttacagtgg gagtatctat 180

tacactccgt ccctcaagag tcgacttacc atatcagtgg acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tatattactg tgcgagtttg 300

gattcctggg gatctaaccg tgactactgg ggccagggaa ccctggtcac cgtctcgagt 360

ggaggtggtg gatccgaaat tgtgttgacg cagtctccag ccaccctgtc tttgtctcca 420

ggggaaagag ccaccctctc ctgcagggcc agtcagagtg ttagcagcta cttagcctgg 480

taccaacaga aacctggcca ggctcccagg ctcctcatct atgatgcatc caacagggcc 540

actggcatcc cagccaggtt cagtggcagt gggtctggga cagacttcac tctcaccatc 600

agcagcctag agcctgaaga ttttgcagtt tattactgtc agcagcgtag caactggccg 660

ctcactttcg gcggagggac caaggtggag attaag 696

<210> 3

<211> 998

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Asp Tyr Arg Gly Tyr Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Ile Val Leu Thr

130 135 140

Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

145 150 155 160

Ser Cys Arg Ala Ser Gln Ser Val Ile Asn Tyr Leu Ala Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn

180 185 190

Arg Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

210 215 220

Tyr Tyr Cys Gln Gln Arg Arg Asn Trp Pro Leu Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

260 265 270

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr

275 280 285

Thr Phe Thr Asp Phe Glu Met His Trp Val Arg Gln Ala Pro Gly Gln

290 295 300

Gly Leu Glu Trp Met Gly Asp Ile Asp Pro Gly Thr Gly Asp Thr Ala

305 310 315 320

Tyr Asn Leu Lys Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser

325 330 335

Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr

340 345 350

Ala Val Tyr Tyr Cys Ala Leu Gly Ala Phe Val Tyr Trp Gly Gln Gly

355 360 365

Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly Ser Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Val Val Met Thr Gln Ser

385 390 395 400

Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys

405 410 415

Arg Ser Ser Gln Ser Leu Ala Asn Ser Tyr Gly Asn Thr Tyr Leu Ser

420 425 430

Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gly

435 440 445

Ile Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

450 455 460

Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp

465 470 475 480

Val Gly Val Tyr Tyr Cys Leu Gln Gly Thr His Gln Pro Tyr Thr Phe

485 490 495

Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg

500 505 510

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

515 520 525

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

530 535 540

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

545 550 555 560

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser

565 570 575

Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg

580 585 590

Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg

595 600 605

Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp

610 615 620

Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

625 630 635 640

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

645 650 655

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

660 665 670

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

675 680 685

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

690 695 700

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

705 710 715 720

Met Gln Ala Leu Pro Pro Arg Glu Gly Arg Gly Ser Leu Leu Thr Cys

725 730 735

Gly Asp Val Glu Glu Asn Pro Gly Pro Met Thr Asn Lys Cys Leu Leu

740 745 750

Gln Ile Ala Leu Leu Leu Cys Phe Ser Thr Thr Ala Leu Ser Gln Val

755 760 765

Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser His Thr Leu

770 775 780

Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Gly Tyr

785 790 795 800

Tyr Trp Ser Trp Thr Arg Gln His Pro Gly Met Gly Leu Glu Trp Ile

805 810 815

Gly Tyr Ile Ser Tyr Ser Gly Ser Ile Tyr Tyr Thr Pro Ser Leu Lys

820 825 830

Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

835 840 845

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

850 855 860

Ser Leu Asp Ser Trp Gly Ser Asn Arg Asp Tyr Trp Gly Gln Gly Thr

865 870 875 880

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr

885 890 895

Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

900 905 910

Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln

915 920 925

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn

930 935 940

Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

945 950 955 960

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

965 970 975

Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu Thr Phe Gly Gly Gly

980 985 990

Thr Lys Val Glu Ile Lys

995

<210> 4

<211> 2994

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaattctat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gaggagaagt 300

gactacaggg gctactacgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 360

tcaggcagta ctagcggtgg tggctccggg ggcggttccg gtgggggcgg cagcagcgaa 420

attgtgttga cacagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc 480

tcctgcaggg ccagtcagag tgttatcaac tacttagcct ggtaccaaca gaaacctggc 540

caggctccca ggctcctcat ctatgatgca tccaacaggg cctctggcat cccagccagg 600

ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 660

gattttgcag tttattactg tcagcagcgt cgcaactggc cgctcacttt cggcggaggg 720

accaaggtgg agatcaaagg aggtggtgga tccggaggtg gtggctccgg aggtggtgga 780

tcccaggttc agctggtgca gtctggagct gaggtgaaga agcctggggc ctcagtgaag 840

gtctcctgca aggcttctgg ttacaccttt accgactttg aaatgcactg ggtgcgacag 900

gcccctggac aagggcttga gtggatggga gatattgatc ctggaactgg tgatactgcc 960

tacaatctga agttcaaggg cagagtcacc atgaccacag acacatccac gagcacagcc 1020

tacatggagc tgaggagcct gaggtctgac gacacggccg tgtattactg tgcgttgggg 1080

gcctttgttt actggggcca gggaaccctg gtcaccgtct cctcaggcag tactagcggt 1140

ggtggctccg ggggcggttc cggtgggggc ggcagcagcg atgttgtgat gactcagtct 1200

ccactctccc tgcccgtcac ccctggagag ccggcctcca tctcctgcag gtctagtcag 1260

agtcttgcaa acagttatgg gaacacctat ttgtcttggt acctgcagaa gccagggcag 1320

tctccacagc tcctgatcta tgggatttcc aacagatttt ctggggtccc tgacaggttc 1380

agtggcagtg gatcaggcac agattttaca ctgaaaatca gcagagtgga ggctgaggac 1440

gttggggttt attactgctt acaaggtaca catcagccgt acacgtttgg ccaggggacc 1500

aagctggaga tcaaaaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 1560

gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 1620

catacccggg gtcttgactt cgcctgcgat atctacattt gggcccctct ggctggtact 1680

tgcggggtcc tgctgctttc actcgtgatc actctttact gtaggagtaa gaggagcagg 1740

ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1800

taccagccct atgccccacc acgcgacttc gcagcctatc gctcccgcgt gaaattcagc 1860

cgcagcgcag atgctccagc ctacaagcag gggcagaacc agctctacaa cgaactcaat 1920

cttggtcgga gagaggagta cgacgtgctg gacaagcgga gaggacggga cccagaaatg 1980

ggcgggaagc cgcgcagaaa gaatccccaa gagggcctgt acaacgagct ccaaaaggat 2040

aagatggcag aagcctatag cgagattggt atgaaagggg aacgcagaag aggcaaaggc 2100

cacgacggac tgtaccaggg actcagcacc gccaccaagg acacctatga cgctcttcac 2160

atgcaggccc tgccgcctcg ggagggaagg ggttctctcc tgacctgcgg cgatgtggag 2220

gagaaccccg gacccatgac caacaagtgt ctcctccaaa ttgctctcct gttgtgcttc 2280

tccactacag ctctttccca ggtgcagcta cagcagtcgg gcccaggact ggtgaagcct 2340

tcacacaccc tgtccctcac ctgcactgtc tctggtggct ccatcagcag tggtggttac 2400

tactggagtt ggacccgtca gcacccaggg atgggcctgg agtggattgg atacatctct 2460

tacagtggga gtatctatta cactccgtcc ctcaagagtc gacttaccat atcagtggac 2520

acgtctaaga accagttctc cctgaagctg agctctgtga ctgccgcgga cacggccgta 2580

tattactgtg cgagtttgga ttcctgggga tctaaccgtg actactgggg ccagggaacc 2640

ctggtcaccg tctcgagtgg aggtggtgga tccgaaattg tgttgacgca gtctccagcc 2700

accctgtctt tgtctccagg ggaaagagcc accctctcct gcagggccag tcagagtgtt 2760

agcagctact tagcctggta ccaacagaaa cctggccagg ctcccaggct cctcatctat 2820

gatgcatcca acagggccac tggcatccca gccaggttca gtggcagtgg gtctgggaca 2880

gacttcactc tcaccatcag cagcctagag cctgaagatt ttgcagttta ttactgtcag 2940

cagcgtagca actggccgct cactttcggc ggagggacca aggtggagat taag 2994

<210> 5

<211> 2181

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaattctat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gaggagaagt 300

gactacaggg gctactacgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 360

tcaggcagta ctagcggtgg tggctccggg ggcggttccg gtgggggcgg cagcagcgaa 420

attgtgttga cacagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc 480

tcctgcaggg ccagtcagag tgttatcaac tacttagcct ggtaccaaca gaaacctggc 540

caggctccca ggctcctcat ctatgatgca tccaacaggg cctctggcat cccagccagg 600

ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 660

gattttgcag tttattactg tcagcagcgt cgcaactggc cgctcacttt cggcggaggg 720

accaaggtgg agatcaaagg aggtggtgga tccggaggtg gtggctccgg aggtggtgga 780

tcccaggttc agctggtgca gtctggagct gaggtgaaga agcctggggc ctcagtgaag 840

gtctcctgca aggcttctgg ttacaccttt accgactttg aaatgcactg ggtgcgacag 900

gcccctggac aagggcttga gtggatggga gatattgatc ctggaactgg tgatactgcc 960

tacaatctga agttcaaggg cagagtcacc atgaccacag acacatccac gagcacagcc 1020

tacatggagc tgaggagcct gaggtctgac gacacggccg tgtattactg tgcgttgggg 1080

gcctttgttt actggggcca gggaaccctg gtcaccgtct cctcaggcag tactagcggt 1140

ggtggctccg ggggcggttc cggtgggggc ggcagcagcg atgttgtgat gactcagtct 1200

ccactctccc tgcccgtcac ccctggagag ccggcctcca tctcctgcag gtctagtcag 1260

agtcttgcaa acagttatgg gaacacctat ttgtcttggt acctgcagaa gccagggcag 1320

tctccacagc tcctgatcta tgggatttcc aacagatttt ctggggtccc tgacaggttc 1380

agtggcagtg gatcaggcac agattttaca ctgaaaatca gcagagtgga ggctgaggac 1440

gttggggttt attactgctt acaaggtaca catcagccgt acacgtttgg ccaggggacc 1500

aagctggaga tcaaaaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 1560

gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 1620

catacccggg gtcttgactt cgcctgcgat atctacattt gggcccctct ggctggtact 1680

tgcggggtcc tgctgctttc actcgtgatc actctttact gtaggagtaa gaggagcagg 1740

ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1800

taccagccct atgccccacc acgcgacttc gcagcctatc gctcccgcgt gaaattcagc 1860

cgcagcgcag atgctccagc ctacaagcag gggcagaacc agctctacaa cgaactcaat 1920

cttggtcgga gagaggagta cgacgtgctg gacaagcgga gaggacggga cccagaaatg 1980

ggcgggaagc cgcgcagaaa gaatccccaa gagggcctgt acaacgagct ccaaaaggat 2040

aagatggcag aagcctatag cgagattggt atgaaagggg aacgcagaag aggcaaaggc 2100

cacgacggac tgtaccaggg actcagcacc gccaccaagg acacctatga cgctcttcac 2160

atgcaggccc tgccgcctcg g 2181

<210> 6

<211> 727

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Asp Tyr Arg Gly Tyr Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly

115 120 125

Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Ile Val Leu Thr

130 135 140

Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

145 150 155 160

Ser Cys Arg Ala Ser Gln Ser Val Ile Asn Tyr Leu Ala Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn

180 185 190

Arg Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

210 215 220

Tyr Tyr Cys Gln Gln Arg Arg Asn Trp Pro Leu Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

260 265 270

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr

275 280 285

Thr Phe Thr Asp Phe Glu Met His Trp Val Arg Gln Ala Pro Gly Gln

290 295 300

Gly Leu Glu Trp Met Gly Asp Ile Asp Pro Gly Thr Gly Asp Thr Ala

305 310 315 320

Tyr Asn Leu Lys Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser

325 330 335

Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr

340 345 350

Ala Val Tyr Tyr Cys Ala Leu Gly Ala Phe Val Tyr Trp Gly Gln Gly

355 360 365

Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly Ser Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Val Val Met Thr Gln Ser

385 390 395 400

Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys

405 410 415

Arg Ser Ser Gln Ser Leu Ala Asn Ser Tyr Gly Asn Thr Tyr Leu Ser

420 425 430

Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gly

435 440 445

Ile Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

450 455 460

Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp

465 470 475 480

Val Gly Val Tyr Tyr Cys Leu Gln Gly Thr His Gln Pro Tyr Thr Phe

485 490 495

Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg

500 505 510

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

515 520 525

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

530 535 540

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

545 550 555 560

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser

565 570 575

Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg

580 585 590

Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg

595 600 605

Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp

610 615 620

Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

625 630 635 640

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

645 650 655

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

660 665 670

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

675 680 685

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

690 695 700

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

705 710 715 720

Met Gln Ala Leu Pro Pro Arg

725

Claims

1. The single-chain antibody for resisting TIM3 is characterized in that the amino acid sequence of the single-chain antibody for resisting TIM3 is shown as SEQ ID No. 1.

2. A nucleic acid encoding the single chain antibody to TIM3 of claim 1.

3. The nucleic acid according to claim 2, wherein the nucleic acid has the nucleotide sequence shown in SEQ ID No. 2.

4. A fusion protein comprising, in order, an antigen binding domain, a transmembrane domain, and an intracellular signaling domain; the amino acid sequence of the intracellular signaling domain comprises the amino acid sequence of the single chain antibody against TIM3 of claim 1.

5. The fusion protein of claim 4, wherein said antigen binding domain comprises a single chain antibody against CD44 and/or a single chain antibody against CD133 and said intracellular signaling domain comprises a single chain antibody against TIM 3.

6. The fusion protein of claim 4, wherein the fusion protein has an amino acid sequence shown in SEQ ID No. 3.

7. A fusion nucleic acid encoding the fusion protein of claim 4.

8. The fusion nucleic acid of claim 7, wherein the fusion nucleic acid has the nucleotide sequence shown in SEQ ID No. 4.

9. An expression vector having inserted therein an expression cassette comprising a first nucleic acid segment encoding an antigen binding molecule and a second nucleic acid segment encoding an intracellular signaling molecule comprising a single chain antibody against TIM3 of claim 1, wherein an IRES element or 2A peptide coding sequence is inserted between the first and second nucleic acid segments.

10. The expression vector of claim 9, wherein the expression cassette is the fusion nucleic acid of claim 7.

11. A cell expressing a chimeric antigen receptor comprising the single chain antibody against TIM3 of claim 1, wherein the cell is obtained by transfecting a host cell with the expression vector of claim 9.

12. The cell of claim 11, wherein the host cell is a T cell.

13. Use of a single chain antibody against TIM3 according to claim 1, a nucleic acid according to claim 2, a fusion protein according to claim 4, a fusion nucleic acid according to claim 7, an expression vector according to claim 9, a cell expressing a chimeric antigen receptor according to claim 11 or 12 for the preparation of a medicament for the treatment of glioma.

14. The use according to claim 13, wherein the glioma is a brain glioma.

15. A pharmaceutical composition comprising the cells of claim 11 or 12 expressing an anti-TIM 3 chimeric antigen receptor as an active ingredient.